The glucocorticoid receptor (GR) is recovered from hormone-free cells in a heterocomplex with the molecular chaperone hsp90, which is required to produce the proper folding state for steroid binding. GR⅐hsp90 heterocomplexes are formed by a multiprotein system that appears to exist in all eukaryotic cells. Recently, we have reconstituted a receptor⅐hsp90 heterocomplex assembly system with purified rabbit hsp90 and hsp70 and bacterially expressed human p23 and p60. We have shown that hsp90, p60, and hsp70 form an hsp90⅐p60⅐hsp70 complex that converts the GR from a non-steroid binding to a steroid binding form ( In the current work, we show that the purified rabbit hsp70 utilized in prior studies is contaminated with a small amount of the rabbit DnaJ homolog hsp40. Elimination of the hsp40 from the purified GR⅐hsp90 assembly system reduces assembly activity, and the activity is restored by addition of the purified yeast DnaJ homolog YDJ-1. hsp40 is a component of the hsp90⅐p60⅐hsp70 foldosome complex isolated from reticulocyte lysate with antibody against p60. Under conditions that promote binding of p23 to hsp90 (elevated temperature, ATP, Nonidet P-40, molybdate), a five-membered (p23⅐hsp90⅐p60⅐hsp70⅐hsp40) complex of chaperone proteins is formed in reticulocyte lysate or from purified proteins. The hsp40-free, purified assembly system has a modest level of assembly activity that is maximally potentiated by YDJ-1 when it is present at about one-twentieth the concentration of hsp70. Although hsp40 is not in the final GR⅐hsp90 heterocomplex isolated from L cell cytosol, it is in the GR⅐hsp90 heterocomplex assembled in reticulocyte lysate. We conclude that hsp40 is a component of the multiprotein hsp90-based chaperone system where it potentiates GR⅐hsp90 heterocomplex assembly.

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The majority of anticancer drugs in clinical use have their utility limited by their toxicity to all proliferating cells, including normal cells and the inability to exert their effect on all of the tumor cells. Novel cytotoxic agents continue to be developed with unique mechanisms of action meant to provide increased targeting, however, many of these compounds still lack absolute tumor selectivity and continue to be limited in their therapeutic utilization due to off-target effects. Antibody drug conjugates (ADCs) have been design to bind to specific epitopes on the surface of tumor cells and have offered an alternative method to target tumor cells in an effort to reduce associated toxicities. Although highly selective, very few antibody drug conjugates are therapeutically useful since they only achieve modest cellular uptake and limited cell killing activity. Here we describe the development of a novel targeting platform using phospholipid ether (PLE) molecules to provide tumor cell specific targeting. These molecules known as phospholipid drug conjugates (PDCs) show high specificity for tumor cells; 6 to 30-fold increase in targeting tumor cells over normal tissue, result in up to 20% of exposed compound entering in to the tumor cells (as compared to < 1% of an ADC), and show a range of compounds can be conjugated to them. We have tested the uptake of our PDCs across over 80 different tumor cell lines and have shown consistent preferential uptake by tumor cells versus normal cells even when in co-culture. This could result in an improvement of the therapeutic index for many compounds. Citation Format: Jarrod Longcor, Maria Banach, Randall Hoover. Phospholipid drug conjugates show specificity for a broad range of tumor cells and provides a novel approach for targeted or precision therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-063.

Background: Multiple myeloma remains an incurable disease. Treatment with immunomodulators and proteasome inhibitors have provided significant benefits to patients but they still relapse. The introduction of new treatments like daratumumab have provided further benefit. However, patients continue to experience issues and relapse. CLR 131 is a novel radioiodinated therapeutic that exploits the selective uptake and retention of phospholipid ethers (PLEs) by malignant cells to provide targeted delivery of iodine-131 directly to tumor cells. This study evaluates the effect of fractionated injections of CLR 131 in OPM-2 tumor bearing mice in comparison to equivalent doses of CLR 131 and the standard dosing of bortezomid. Methods: The OPM-2 cell line (human multiple myeloma) was purchased from American Type Culture Collection (ATCC, Rockville, MD) and maintained in McCoy's 5a media supplemented with 10% fetal bovine serum. Female CB17 SCID mice approximately 5-7 weeks of age were injected subcutaneously with 1x107 viable cells (in ~100 µL Dulbecco's PBS) into the right flank. The study was initiated when tumor size had reached a pre-determined size (approximately 150-200 mm3). The mice were given potassium iodide at a concentration of 0.1% in their drinking water to block possible free iodide in the drug formulation three days prior to injection and continuing through two weeks post-injection. Results: All treatment groups showed a reduction in tumor growth with the fractionated dose of CLR 131 producing the greatest inhibition. The control group showed exponential growth throughout the study, increasing in volume by an average 11 fold from baseline. This compared with a 3.5 fold increase from baseline in the fractionated dose group and 7.5 fold for the bortezomid group. Conclusions: The results of the study indicated that a fractioned injection of CLR 131 (2 injections of ~50 µCi) on human multiple myeloma (OPM-2) tumor bearing model showed a significant inhibition of tumor growth as well as a survival benefit. While a single injection of CLR 131 at either ~50 uCi and ~100 uCi were effective, there was an important improvement with by using fractionated dosing. The implication is that in this clinically predictive in vivo model CLR 131, which is currently in a Phase 2 clinical trial for multiple myeloma, might show improved clinical efficacy in patients if a fractionated dose is utilized. Citation Format: Jarrod Longcor, Maria Banach, John Friend. Efficacy of fractionated injections of CLR 131 in a OPM-2 SCID nude mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-279.